Journal of Plant Physiology 171 (2014) 576–586

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Physiology

Genotypic response of detached leaves versus intact plants for chlorophyll fluorescence parameters under high temperature stress in wheat Dew Kumari Sharma a,c,∗ , Juan Olivares Fernández b , Eva Rosenqvist c , Carl-Otto Ottosen d , Sven Bode Andersen a a Department of Plant and Environmental Sciences, Section of Plant and Soil Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark b Universidad Politécnica de Madrid, Escuela Técnica Superior de Ingenieros Agrónomos, Avenida Complutense s/n, 28040 Madrid, Spain c Department of Plant and Environmental Sciences, Section for Crop Sciences, University of Copenhagen, Hojbakkegaard Allé 9, 2630 Taastrup, Denmark d Department of Food Science, Aarhus University, Kirstinebjergvej 10, 5792 Aarslev, Denmark

a r t i c l e

i n f o

Article history: Received 13 May 2013 Received in revised form 10 September 2013 Accepted 15 September 2013 Available online 20 March 2014 Keywords: Chlorophyll fluorescence Detached leaves Heat stress Temperature Wheat

a b s t r a c t The genotypic response of wheat cultivars as affected by two methods of heat stress treatment (treatment of intact plants in growth chambers versus treatment of detached leaves in test tubes) in a temperature controlled water bath were compared to investigate how such different methods of heat treatment affect chlorophyll fluorescence parameters. A set of 41 spring wheat cultivars differing in their maximum photochemical efficiency of photosystem (PS) II (Fv /Fm ) under heat stress conditions was used. These cultivars were previously evaluated based on the heat treatment of intact plants. The responses of the same cultivars to heat stress were compared between the two methods of heat treatment. The results showed that in detached leaves, all of the fluorescence parameters remained almost unaffected in control (20 ◦ C at all durations tested), indicating that the detachment itself did not affect the fluorescence parameters. In contrast, heat induced reduction in the maximum photochemical efficiency of PSII of detached leaves occurred within 2 h at 40 ◦ C and within 30 min at 45 ◦ C, and the response was more pronounced than when intact plants were heat stressed for three days at 40 ◦ C. The proportion of total variation that can be ascribed to the genetic differences among cultivars for a trait was estimated as genetic determination. During heat treatment, the genetic determination of most of the fluorescence parameters was lower in detached leaves than in intact plants. In addition, the correlation of the cultivar response in intact plants versus detached leaves was low (r = 0.13 (with expt.1) and 0.02 with expt.2). The most important difference between the two methods was the pronounced difference in time scale of reaction, which may indicate the involvement of different physiological mechanisms in response to high temperatures. Further, the results suggest that genetic factors associated with cultivar differences are different for the two methods of heat treatment. © 2013 Elsevier GmbH. All rights reserved.

Introduction

Abbreviations: (1 − Vj )/Vj , variable fluorescence at J step in the fast fluorescence transient curve (OJIP curve); Area, relative area above the OJIP curve from Fm ; Fm , maximum fluorescence; Fo , minimum fluorescence; Fv , variable fluorescence; Fv /Fm , maximum photochemical efficiency of PSII; Fv /Fo , maximum primary yield of photochemistry of PSII; PI, performance index; PPFD, photosynthetic photon flux density; PSII, photosystem II; RC/ABS, active reaction centers per absorbance; TFm , time to reach Fm . ∗ Corresponding author at: Department of Plant and Environmental Sciences, Section of Plant and Soil Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. Tel.: +45 35 33 32 10. E-mail address: [email protected] (D.K. Sharma). 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.09.025

Wheat (Triticum aestivum L.) is one of the most important food crops, cultivated on more than 216 million hectares of farmland worldwide with overall production of 651 million tons (FAO, 2012). Heat stress as a result of climate change may negatively affect wheat grain yields (Ortiz et al., 2008). The effect of heat stress on photosynthetic performance was foreseen by the discovery of thermal instability of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Feller et al., 1998; Sayed, 2003) and inhibition of electron transport in photosystem II (PSII) (Haldimann and Feller, 2005; Mathur et al., 2011). In addition to agronomic and other management strategies, improvement of heat tolerance in wheat through breeding and

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molecular tools is in progress (Farooq et al., 2011). However, the large diversity of wheat cultivars combined with polyploidy and gene redundancy makes it difficult to identify desirable genotypes (Dong et al., 2009; Farooq et al., 2011). Therefore, physiological and biochemical screening techniques are desirable to complement phenotypic measurements in order to increase the selection efficiency (Ibrahim and Quick, 2001; Reynolds et al., 1994). Traits previously studied for screening of wheat for heat tolerance include cell membrane thermo stability (Farooq et al., 2011; Ibrahim and Quick, 2001), cellular respiration (Farooq et al., 2011), canopy temperature depression (Ali et al., 2010; Reynolds et al., 1994) and individual kernel weight (Sharma et al., 2008). Most of these methods require long measurement time or involve complex laboratory techniques. In addition, evaluation of heat tolerance under field conditions based on yield parameters is a slow process, influenced by many factors. The multiple environmental factors affecting field trials complicate phenotyping of material adapted to different growing conditions, because the response to a specific stress confounds the effects of plant adaptation. In stress physiology, chlorophyll fluorescence is an important non-invasive technique in assessing and quantifying damage to the leaf photosynthetic apparatus, particularly PSII activity in response to environmental stresses (Baker and Rosenqvist, 2004; Maxwell and Johnson, 2000). However, this method also has some common pitfalls associated with the measurements and analysis (Logan et al., 2007). Thus, due care should be taken to create uniform conditions including the use of uniform illumination of sampling leaves during the heat treatment, plants of equal developmental stage, etc., all factors that may influence the fluorescence measurements, when different genotypes of diverse origin are being treated and compared (Sharma et al., 2012). We have used Fv /Fm as the selection criterion for heat stress tolerance (Sharma et al., 2012). As a measure of the maximum photochemical efficiency of PSII (Fv /Fm ) (Baker and Rosenqvist, 2004), usually any decrease in Fv /Fm results in a corresponding decrease in the maximum apparent quantum yield of the photosynthetic light response curve measured by gas exchange at ambient CO2 concentration (Ögren and Sjöström, 1990) as well as when measured as oxygen evolution at saturating CO2 (Demmig and Björkman, 1987). Changes in Fv /Fm thus may have a direct effect on the carbon gain of the plant, especially when light is a limiting factor in the photosynthesis. In a heat pulse treatment (50 ◦ C for 20–40 s), the reduction in Fv /Fm has been reported to represent the loss of PSII capacity rather than loss of maximum quantum yield of PSII (Tóth et al., 2005), and the destruction of the manganese cluster by a heat pulse affected the oxygen evolution but had a small effect on the quantum yield of PSII (Tóth et al., 2007). The quantitative meaning of Fv /Fm is based on the QA model (Duysens and Sweers, 1963), with the assumption that change in fluorescence reflects the changes in the redox state of QA in PSII, and this parameter has been used in numerous stress physiological studies to relate the maximum quantum yield of PSII photochemistry. Recently, the QA plus light-induced conformational change model have been proposed to better understand the quantitative meaning of Fv /Fm (Schansker et al., 2013). Some fluorometers also calculate other parameters from the fast phase of the fluorescence induction curve, also called the OJIP curve, used for the JIP-test. They are e.g. the maximum primary yield of photochemistry of PSII (Fv /Fo ), active reaction centers per absorbance (RC/ABS) and the performance index (PI), while time to reach Fm (TFm) is a descriptive parameter estimating the time to reach maximum fluorescence, (1 − Vj )/Vj a fluorescence transient curve-derived parameter related to the probability with which a PSII trapped electron is transferred from QA to QB and Area is the relative area above the OJIP curve (Stirbet and Govindjee, 2011; Strasser et al., 2004). Many of the JIP-test parameters are interdependent (Strasser et al., 2004) and it is not clear yet how the overall

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effect of heat stress on carbon gain in plants is reflected by these parameters. Under field conditions, the effects of heat stress on plants are often confounded with other factors such as drought and light. Apart from the high cost of conducting yield trials under various stress conditions, the difficulty in separating such effects of different stresses and stress periods complicates the screening protocol for single stress factors. Treating intact plants in controlled environments is a viable option, but limitations in capacity and resources restrict the number of genotypes to be evaluated. Therefore, cheaper, faster and easier screening methods to measure heat tolerance capacity in large numbers of genotypes would be valuable for the selection of wheat cultivars. In general, many plant physiological responses to elevated temperatures have been investigated by in vitro studies with the use of detached leaf segments or isolated organelles. Some studies have been performed using a water bath to heat stress detached leaves in tubes, demonstrating the influence of elevated temperature on chlorophyll fluorescence parameters (Havaux, 1993; Mathur et al., 2011). This detached leaf method not only allows an excellent isolation of heat stress from the influence of other abiotic factors such as light, water and nutrient supply, but it also provides an opportunity for fast evaluation of a large number of plant samples. However, it is still not known if a detached leaf system can also be used to select plants for their heat tolerance. The main aim of the present study was to test the genotypic response of wheat cultivars to the effect of heat treatment on the detached leaves versus intact plants using the chlorophyll fluorescence technique of stress detection. Materials and methods Plant material The plant material consisted of 41 spring wheat cultivars, previously shown, using intact plants, to differ in their heat tolerance, evaluated as their maximum photochemical efficiency of photosystem II (PSII) (Fv /Fm ) by chlorophyll a fluorescence (Sharma et al., 2012). These cultivars belong to different wheat growing regions of the world (Sharma et al., 2012). Growing conditions Single seeds were sown in pots (11 cm diameter; 0.59 L) filled with peat (Pindstrup 2, Pindstrup Mosebrug A/S, Ryomgaard, Denmark). The plants were grown in a greenhouse under long day conditions (16/8 h day/night) with air temperature of 15 ± 3 ◦ C during the day and 12 ± 3 ◦ C during the night, 50–70% relative humidity and ambient CO2 concentration. Supplementary light of 60 ␮mol photons m−2 s−1 was provided from high-pressure sodium lamps (SON-T Agro, 600W, Phillips, Eindhoven, The Netherlands) in the evening time, so as to maintain the photoperiod of 16 h. Plants were fertilized with a nutrient solution consisting of: N (185 mg L−1 ), P (27 mg L−1 ), K (171 mg L−1 ), Mg (20 mg L−1 ) and full micronutrients. The plants were grown under these conditions until the phenotypic stage ranged from late stem elongation to the inflorescence emergence stage based on BBCH identification keys of wheat (Lancashire et al., 1991). Experimental set up Two experiments (separated in time) were conducted with detached leaf treatment in a water bath. Each experiment had three complete randomized blocks and the blocks were separated in time by one week in experiment 1 and two weeks in experiment 2. In experiment 1, four plants of each cultivar were used in each block (3 blocks × 4 plants = 12 plants per cultivar). In experiment 2,

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three plants of each cultivar were used in each block (3 blocks × 3 plants = 9 plants per cultivar).

prior to heat treatment were considered as untreated controls i.e. day 0 or 0 min.

Experiment 1

Statistical analysis

Three days prior to the treatment, plants were moved to a climate chamber (Prepan Coldstore Contracting, Denmark) to keep the plants under more controlled condition as follows: photoperiod 16/8 h; light/dark, photosynthetic photon flux density (PPFD) 70–80 ␮mol photons m−2 s−1 , air temperature 20 ◦ C, relative humidity 70% and CO2 400 ␮L L−1 with regular irrigation. For heat treatment, a 5–8 cm long piece of the penultimate leaf (sampling leaf) was cut off from the plant and placed in a borosilicate glass tube of 20 mm diameter (Ref. 617300, Deltalab S.L., Barcelona, Spain). A few drops of distilled water were added into the glass tube to avoid evaporative loss from leaves during the heat treatment. Then, glass tubes with leaf samples were placed in a holder and capped with polypropylene caps (Deltalab S.L., Barcelona, Spain) and immersed in a water bath controlled by a thermostat (Lauda E100, GmbH and Co. K.G., Lauda-Königshofen, Germany). The temperature inside the glass tubes was measured by a thermometer (Evolution N9008, COMARK Ltd., Norwich, United Kingdom) with thin thermocouples that could reach to the bottom of the glass tube. The heat treatment was performed in the darkness to isolate the heat effect from the confounding effect of light. The temperature treatments consisted of 20 ◦ C (control) and 40 ◦ C (stress) for 15, 30, 60 and 120 min duration. A higher temperature of 45 ◦ C was also tested for 15 and 30 min duration. Separate leaf samples of each plant were used for different time points.

The analysis of the experimental data was done using Statistical Analysis System (SAS version 9.2 (SAS Institute Inc., Cary, NC, USA). To analyze the data correctly, transformation was required as the raw data did not show uniform normal distribution owing to an upper limit in most of these parameters (e.g. 0.85 for Fv /Fm ). Therefore, the raw data of each fluorescence parameters were transformed using power transformation (Box and Cox, 1964). The transformation used the formula (  − 1)/ with  = 11 for Fv /Fm ,  = 5 for (1 − Vj )/Vj ,  = 3 for Fv /Fo and RC/ABS,  = 1 for Area and PI, and  = 0.3 for TFm . These values of  were initially determined from plots of residuals from analysis of variance to produce residuals with approximately uniform variation over the entire spectrum of model values. Fluorescence parameters from each experiment were subsequently analyzed by two-way ANOVA (PROC GLM) with main effects of blocks and cultivars. An F-test was done to determine whether differences between cultivars were significantly different. Means of each cultivar over blocks were calculated and later back-transformed to use for the figures and tables. For both the control and heat treatments, cultivar variance components (G2 ) and error variance components (E2 ) were estimated on the transformed scale by the PROC MIXED procedure (SAS) with blocks as fixed and cultivars as random effects. These variance components were used to calculate genetic determination (i.e. the proportion of total variation that is due to the genotype) of the chlorophyll fluorescence parameters with the formula, GD = G2 /(G2 + E2 ) in order to estimate relative importance of differences among cultivars (further discussed in Sharma et al., 2012). The PROC MIXED was also used for each treatment time point separately to obtain genetic determination of chlorophyll fluorescence parameters under each treatment. Further, the fluorescence transient curves, commonly called OJIP curves, from each measurement were approximated with a smoothing spline (Jspline + java library) and their first order derivative were aligned by adjustment of the time scale as described by (James, 2007). The aligned first derivatives were used to calculate average curves for each treatment and numerically integrated to obtain average OJIP curves for each treatment.

Experiment 2 The main aim of this experiment was test the reproducibility of cultivar response in two repeated experiments with detached leaf treatment in the water bath. Therefore, this experiment was conducted in a similar way as e experiment 1 except that only one treatment combination i.e. 40 ◦ C for 30 min was tested. For this experiment, a different batch of plants of the same 41 cultivars was grown and this experiment was separated from experiment 1 in time. This experiment was conducted simultaneously with the intact plant method described previously (Sharma et al., 2012), to which the results of the present study were compared to understand how different methods of heat treatment affect cultivar response and genetic determination for chlorophyll fluorescence parameters.

Results

Chlorophyll fluorescence measurements

Temperature, duration and cultivar-dependant effects on detached leaves (experiment 1)

The treated leaves were removed from the glass tubes after the pre-set duration of treatment and clipped with dark adaptation clips (Hansatech Instrument, King’s Lynn, England) for 30 min at room temperature (22 ◦ C). To avoid leaf desiccation during the dark adaptation period, the samples were kept in a plastic tray lined with moist tissue paper and covered with a plastic bag. Chlorophyll a fluorescence parameters were measured on the adaxial leaf surface by a Handy PEA (Hansatech Instrument, King’s Lynn, England) using a PPFD of 3000 ␮mol photons m−2 s−1 as saturating flash for the duration of 1 s. The OJIP curves (fast fluorescence transients) and the associated fluorescence parameters such as Fv /Fm (maximum quantum efficiency of PSII), Fv /Fo (maximum primary yield of photochemistry of PSII), TFm (time to reach maximum fluorescence, Fm ), Area (relative area above the fluorescence transients curve from Fm ), RC/ABS (active reaction centers per absorbance), (1 − Vj )/Vj , (variable fluorescence at J step in the fluorescence transient curve), and PI (performance index) were recorded (Strasser et al., 2004; Stirbet and Govindjee, 2011). Measurements taken

All of the fluorescence parameters remained almost unaffected at all durations in the control (20 ◦ C) (Fig. 1A–G). The Fv /Fm declined by 28% after 120 min at 40 ◦ C compared to the control (20 ◦ C). However at 45 ◦ C, the Fv /Fm dropped abruptly by 37% within 15 min, followed by a further decrease at 30 min (47%) as compared to the control (Fig. 1A). Of the total variation in Fv /Fm at 20 ◦ C, around 15% was attributable to genotypic differences, but this genetic determination was reduced at higher temperatures, and the reduction was even more abrupt in 45 ◦ C than in 40 ◦ C (Fig. 2A). At 40 ◦ C for 15 min, all of the cultivars reacted in a similar manner, showing almost no genotypic differences, and the genotypic differences gradually increased with longer durations of treatment. However, the highest genetic determination of 11.7% after 120 min at 40 ◦ C was still lower than in the control (Fig. 2A). The effect of heat stress on Fv /Fo was similar to that of Fv /Fm and was significantly affected by heat stress and duration of treatment at 40 ◦ C (Fig. 1B), but the effect was more pronounced at 45 ◦ C. The genetic variation followed an almost identical pattern as that of

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Fig. 1. Effect of detached leaf method of heat treatment on various chlorophyll fluorescence parameters. Each bar represents mean ± SE calculated from 12 plants each of 41 wheat cultivars. Control (20 ◦ C) and heat stress (40 and 45 ◦ C). The heat stress had a significant (p < 0.0001) effect on all the chlorophyll fluorescence parameters at all the durations tested.

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Fig. 2. Genetic determination (GD) of various chlorophyll fluorescence parameters in detached leaf method of heat treatment. Control (20 ◦ C) and heat stress (40 and 45 ◦ C). The GD ± SE was calculated based on 12 plants each of 41 wheat cultivars.

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Fv /Fm (Fig. 2B), which is expected since Fv /Fm and Fv /Fo are mathematically related, which means that any factor affecting Fv /Fm also affects Fv /Fo (Fig. 1B). The TFm was attained at around 300 ms and remained unaffected by the duration at 20 ◦ C, but showed an interesting trend at elevated temperatures (Fig. 1C). At 40 ◦ C, an increase in TFm was observed at 15 and 30 min of treatment, but decreased again as the duration increased (60 and 120 min). The TFm at 45 ◦ C decreased abruptly within 15 and 30 min duration (Fig. 1C). However, this variation in TFm did not reflect cultivar differences under heat stress conditions, as genetic determination was generally low and did not differ noticeably between control and treated leaves except for 40 ◦ C at 15 min and 45 ◦ C, which dropped suddenly compared to the control (Fig. 2C). Within the first 15 min at 40 ◦ C, the leaves showed a heat shock response in all cultivars. Subsequently, the different cultivars adjusted to the heat stress with different efficiency. However, at 45 ◦ C, the heat stress was too strong for the cultivars to show any genetic difference (Fig. 2C). Similar to other fluorescence parameters, the RC/ABS remained stable in the control but gradually decreased with duration and severity of the stress. The value dropped by 60% at 40 ◦ C for 120 min, while at 45 ◦ C, it drastically decreased by 84% within 30 min as compared to the control (Fig. 1D). The (1 − Vj )/Vj was higher than the control at 15 and 30 min durations at 40 ◦ C (Fig. 1E). The genetic determination of RC/ABS and (1 − Vj )/Vj showed a slightly similar trend at 20 ◦ C for 30 min as compared to the same time points in other parameters (Fig. 2D and E). Further, the reduction in genetic determination at 40 ◦ C for 15 min was less than in other parameters. Heat treatment reduced PI linked to the duration at 40 ◦ C (Fig. 1F). However at 45 ◦ C, there was a sharp decrease in PI value within 15 min with no further decrease at 30 min (Fig. 1F). The genetic determination of PI under heat treatment was around half of the control value (Fig. 2F). The area above the curve at 40 ◦ C increased sharply at 15 and 30 min, followed by gradual decrease at 60 and 120 min (Fig. 1G). At 45 ◦ C, it increased more than the control in 15 min and decreased abruptly at 30 min (Fig. 2G). The genetic component for the area was slightly higher than the control at 40 ◦ C, but it was almost nil at 45 ◦ C (Fig. 2G). Taken together, an interesting pattern was observed in TFm , (1 − Vj /Vj ) and Area at 15 and 30 min at 40 ◦ C, as these parameters increased due to heat treatment, while all other parameters were decreased (Fig. 1C, E and G). This shows that, during heat stress, the maximum PSII capacity (Fv /Fm ) decreases as the population of PSII’s with active reaction center decreases. With fewer active reaction centers, more time is required to reach Fm and if this time extends longer, the acceptor side of the PSII will become activated before Fm is reached. However, such a response was less influenced by genotypic factors under heat stress conditions, as reflected by genetic determination of various fluorescence parameters. The cultivar differences were maintained throughout the treatment at 20 ◦ C (Fig. 2A–G). At 40 ◦ C, leaves exhibited a shock reaction until 15 min, showing no cultivar differences, which slowly changed with time, but the differences were still lower than the control. At 45 ◦ C, the severity of the heat stress did not allow any possibility to observe cultivar-dependent responses for any of the parameters (Fig. 2A–G). Furthermore, in experiment 2, in which detached leaves were treated only at 40 ◦ C for 30 min, we saw a similar trend in fluorescence parameters as in experiment 1, although the values were slightly different (Table 1). Fv /Fm , Fv /Fo , RC/ABS and PI were reduced, and the TFm , (1 − Vj /Vj ) and Area were increased in heat-treated leaves as compared to the control (Table 1). On the other hand, the genetic determination for chlorophyll fluorescence parameters at this time point showed a different trend than in experiment 1, which was comparable to treatment of intact plants (Sharma et al., 2012). However, it is important to note that

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the genetic determination for parameters such as Fv /Fm , Fv /Fo , (1 − Vj /Vj ) and TFm in detached leaves (Table 1) was around half of the value previously found with intact plants (Sharma et al., 2012). For parameters such as RC/ABS, PI, and Area, the genetic determination was similar in control and treated leaves (Table 1). Detached leaves versus intact plant methods Response of cultivars The mean Fv /Fm of each cultivar in the two experiments of detached leaves (experiments 1 and 2) was plotted to examine the correlation of cultivar responses between the repeated experiments in the same treatment combination (i.e. 40 ◦ C for 30 min). A significant positive correlation (r = 0.56***) was found between cultivar means for control leaves from the two experiments (Fig. 3A). Two separate experiments (expt.1 and expt.2) with detached leaves treatment at the same temperature and duration in a water bath (40 ◦ C for 30 min) produced similar genotypic responses to some extent (r = 0.29*) (Fig. 3B), which indicates that genetic differences do exist for detached leaf treatment. However, the effect of cultivar response disappeared between intact plants and detached leaves in both the experiments (Fig. 3C and D). This indicates that the response of cultivars is differentially affected in the two methods. Therefore, selection of cultivars with detached leaf methods may not necessarily operate on the same genetic factors as in intact plants. Fluorescence induction kinetics (OJIP curves) Under control conditions, fluorescence induction kinetics showed the typical three phases, namely OJ, JI and IP, regardless of the duration in both intact plants and detached leaves methods of heat treatment (Fig. 4A and B). Under elevated temperature, the shape of the curves changed depending on the severity of the heat stress in both the methods (Fig. 4C and D). It was clear that the heat stress imposed on detached leaves was more severe than with the intact plants. In intact plants, the effect of heat stress seemed to build up as the duration increased from Day 1 to Day 3 at 40 ◦ C, as indicated by the change in the shape of OJIP curves (Fig. 4C). However, in detached leaves, the change in the shape of the curves was more noticeable with the increase in duration of treatment from 15 to 120 min at 40 ◦ C (Fig. 4D). Heat treatment led to an increase in Fo (O-step), a decrease in Fm (P-step), together with the disappearance of J and I steps depending on the duration of treatment (Fig. 4C and D). Furthermore, heat-induced effects were more apparent in the upper part of the curves (Fig. 4C and D). The additional phase called K step at around 300 ␮s has been reported to occur due to heat stress (Guissé et al., 1995; Lazár and Ilik, 1997). Such a peak was not visible in intact plant (Fig. 4C), but was observed in detached leaves. It was found to be duration-dependant at 40 ◦ C (Fig. 4D), and was more pronounced within 15 min at 45 ◦ C (Fig. 4E). This indicates that the effect of heat stress in the detached leaves in a water bath at 45 ◦ C for 15 min was more severe than the heat stress of 40 ◦ C up to 120 min, which was more severe than the heat stress of 40 ◦ C for 3 days in intact plants in a climate chamber. Relationship between the OJIP parameters and Fv /Fm For comparative evaluation of the effect of heat stress on Fv /Fm versus other OJIP parameters, the different parameters were normalized to their control values and the OJIP parameters were plotted against Fv /Fm (Fig. 5). Fv /Fo showed a close to exponential relationship to Fv /Fm and was mathematically correlated with Fv /Fm (no biological variation in the data), while RC/ABS and PI showed similar patterns as functional relationships (Fig. 5A). The three descriptive parameters (1 − Vj )/Vj , TFm and area showed a considerably more scattered relationship to Fv /Fm (Fig. 5B). However, we noted that 1 s saturating flash was not sufficient to reach the Fm

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Table 1 Chlorophyll fluorescence parameters and their genetic determination in detached leaf method (experiment 2), which was conducted simultaneously with the intact plant method. Control represents measurements prior to the treatment (Day 0) and stress represents heat treatment of detached leaves in a water bath at 40 ◦ C for 30 min. Each value is the average of 9 plants each of 41 wheat cultivars. Parameter

Control Mean ± SE

Fv /Fm Fv /Fo TFm (ms) RC/ABS (1 − Vj )/Vj PI Area (a.u.)

0.84 5.29 298 0.96 0.59 2.98 71,285

± ± ± ± ± ± ±

0.00 0.17 23 0.05*** 0.01*** 0.23*** 4542*

Stress Mean ± SE 0.76 3.28 833 0.75 0.68 1.64 229,469

± ± ± ± ± ± ±

Control GD ± SE (%) 0.01*** 0.13*** 37*** 0.03*** 0.02*** 0.11*** 23,500***

6.68 6.62 19.37 34.46 19.04 34.16 19.90

± ± ± ± ± ± ±

9.18 9.18 9.35 9.27 9.35 9.28 9.35

Stress GD ± SE (%) 13.52 14.34 15.30 39.16 29.81 34.52 23.03

± ± ± ± ± ± ±

4.63 4.72 4.83 6.53 6.32 6.40 5.65

* Significant cultivar difference in the parameter is indicated as P < 0.05. Significant cultivar difference in the parameter is indicated as P < 0.01. *** Significant cultivar difference in the parameter is indicated as P < 0.0001.

**

level at severe treatment (especially at 45 ◦ C, hence these data are excluded and TFm is called TP in Fig. 5). The data include 40 ◦ C treatment data from detached leaves and intact plants, where the latter data are part of the aggregated data of fraction of Fv /Fm > 0.9. A decrease of Fv /Fm from control values of 0.84 to 0.79 (6% decrease from 1.0 to 0.94, which is the strongest decrease found after three days at high temperature in intact plants) is indicated by a dotted line in Fig. 5A. It corresponds to a 26% decrease of Fv /Fo , 31% of RC/ABS and 45% of PI. Discussion Chlorophyll fluorescence parameters were significantly affected by heat stress in both intact plant and detached leaf methods of heat treatment in wheat. We found that the photosynthetic apparatus of wheat leaves is affected much faster in detached

leaves than in intact plants at the same temperature (40 ◦ C). Apparently, the PSII deterioration in detached leaves was more severe within 30 min at 45 ◦ C and within 2 h at 40 ◦ C, than what was observed in intact plants at 40 ◦ C for three days (Sharma et al., 2012). In an earlier study, high temperature-induced changes in photochemical efficiency in wheat have been investigated by treating detached leaves in a water bath (Mathur et al., 2011). It was also reported that detached leaves responded according to the severity of elevated temperature, but showed no effect on photochemical efficiency of PSII at 35 ◦ C followed by a 14% reduction at 40 ◦ C and causing irreversible damage of the oxygen evolving complex in PSII at 45 ◦ C within a duration of 15 min (Mathur et al., 2011). Similarly, the reversible or irreversible damage in PSII capacity depending on the temperature treatment has been reported in pea and potato leaves (Havaux, 1995; Havaux et al., 1991).

Fig. 3. Back-power transformed Fv /Fm of 41 wheat cultivars in intact plant versus detached leaf methods of heat treatment. (A) Cultivar response to 20 ◦ C (control, prior to treatment) between the two methods. (B) Cultivar response to same treatment combination at two experiments with detached leaves. (C and D) Cultivar response to heat stress in detached leaf versus intact plant treatment. Each dot is a cultivar mean from six plants in intact plant and nine plants in detached leaf in experiment 2, and 12 plants in detached leaf in experiment 1.

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Fig. 4. Comparative effect of two methods of heat treatment on chlorophyll a fluorescence transient (OJIP) curves in wheat. The O–J–I–P steps are indicated in (A) and the K-step in (E). (A and C) Intact plant treatments in a climate chamber for three days. (B, D and E) Detached leaf treatments in a water bath. In both the methods, Day 0 is the measurement prior to treatment. Average after alignment of 123 curves (20 ◦ C, intact plants (control)), 246 curves (40 ◦ C, intact plants (heat stress)) and 492 curves (in detached leaves (20 ◦ C (control), 40 ◦ C and 45 ◦ C (heat stress)).

The most important difference between the two methods is the vast difference in the time scale of reaction, which may reflect the involvement of different physiological response mechanisms. In general, plants under natural conditions react to stress either by avoidance or tolerance mechanisms. Some of the heat avoidance mechanisms in grasses involve change in the leaf angle and orientation (Bonos and Murphy, 1999) and evapo-transpirational cooling through stomatal regulation (Reynolds-Henne et al., 2010). Such avoidance processes are vital to reducing damage in key metabolic processes such as photosynthesis. By enclosing the detached leaf in a test tube with a few drops of water, it is exposed to the particular temperature in high air humidity, which prevents cooling both through transpiration and changes in leaf angle and orientation. In addition, intact plants may have some ability to buffer the change in the photosynthetic

apparatus through source-sink balance and internal feedback regulations under stress condition. For instance, in a study with two of the 41 cultivars used in the present study, it was found that the cultivar selected based on high Fv /Fm under heat stress on intact plants also maintained a higher photosynthetic rate (Shanmugam et al., 2013). Further, both of the cultivars accumulated higher sucrose and hexose (glucose and fructose) during heat stress, but hexose, in response to elevated CO2 and heat stress was accumulated only in the more tolerant cultivar having higher Fv /Fm (Shanmugam et al., 2013). This could be a part of differences in internal regulation in carbohydrate balance. Such counteractions or short-term acclimation responses in whole plants toward the stress are not possible when the stress response of cultivars is studied based on the heat treatment of detached leaves in test tubes. For these reasons, it may also be expected that different

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Fig. 5. Relationship between fraction of control values of Fv /Fm and Fv /Fo , TRC/ABS and PI (A) and (1 − Vj )/Vj , the area above the fluorescence curve and TFm (in case of severe heat stress at 45 ◦ C, true TFm was not reached within 1 s, hence TFm = TP ) (B). The dashed line indicates a 6% decrease in Fv /Fm , based on a drop of Fv /Fm from 0.84 to 0.79 found in intact plants. The data originate from both detached leaf and intact plant treatments.

genetic factors among cultivars will affect the results as: (1) an integrated whole plant response, from biochemical processes to stomatal regulation and leaf cooling in intact plants, or (2) biochemical processes in the detached leaf tissues. Further, the change in shape of OJIP curves was affected based on the severity of heat stress, and was found to be more pronounced in detached leaves than in intact plants. The change in OJIP pattern has been proposed to reflect the stepwise flow of energy through PSII at the reaction center level (Strasser et al., 2004). The severe heat stress-induced K step, particularly in detached leaves, may reflect irreversible damage in PSII (Lazár et al., 1999) and destruction of the manganese cluster in the oxygen-evolving complex (Tóth et al., 2005, 2007). In a biophysical study with recovery of PSII following heat pulse of 50 ◦ C for 20 or 40 s, it was found that a large proportion of the manganese cluster was damaged, accompanied by decreased oxygen evolution and Fv /Fm and induction of a K peak (Tóth et al., 2005). Also, a K peak was visible, but following recovery in light for 4 h, it disappeared, which suggests that an accumulation of inactivated PSII reaction centers during heat stress is needed to induce the K step (Tóth et al., 2005). Absence of a K peak in intact plant treatment in the present study indicates that a relatively larger population of inactivated PSII reaction centers have built up in 40 ◦ C within 2 h in detached leaves than in 40 ◦ C for 3 days heat treatment in intact plants. Turnover of damaged components of the PSII, particularly the D1 protein (Baena-Gonzalez and Aro, 2002) and removal of heat impaired PSII reaction centers followed by de novo synthesis of PSII reaction centers (Tóth et al., 2005)

have been reported to be light-mediated processes. Since the heat treatment in intact plants was given for three days in the presence of light, such in situ repair mechanisms in PSII could be faster in intact plants and therefore, accumulation of inactive PSII reaction centers was less in the intact plants than under the heat treatment of detached leaves for a shorter time (within 2 h) in darkness. The primary fluorescence parameter derived from the fluorescence induction curve is the maximum quantum efficiency of PSII, Fv /Fm (Baker and Rosenqvist, 2004). However, a series of other parameters that quantify the shape of the OJIP curve has been developed by Strasser and co-workers and the ease of measuring “make the JIP-test a powerful tool for rapid screening of the ‘vitality’ of plants,” where the term ‘vitality’ integrates “activity and adaptability” of the photosynthetic sample (Strasser et al., 2004). The descriptive parameters (1 − Vj )/Vj , area and TFm do not show any clear relationship to Fv /Fm , but when plotting Fv /Fm to Fv /Fo , they show a close to exponential mathematical relationship (a slight skewness in the residuals of the exponential curve fit and no biological variation in the data, not shown). RC/ABS and PI, follow a similar relationship. Common for these three parameters is that they all show a considerably stronger drop at low stress levels than Fv /Fm . The chosen reference level for comparison (6% decrease of Fv /Fm from 0.84 to 0.79) corresponds to the maximum decrease in Fv /Fm in the experiment with the intact plant method and this is within the range of what is readily recovered within few days at normal temperature (unpublished data). Since a decrease in Fv /Fm has been reported to indicate a corresponding decrease in the maximum quantum yield of the photosynthetic light response curve (Demmig and Björkman, 1987; Ögren and Sjöström, 1990), Fv /Fo , RC/ABS and PI may overestimate any mild heat stress that only has minor effects on the carbon gain of the plant. It also means that any natural variation in control values will be enhanced. When the stress is strong, on the other hand, the three parameters are less sensitive to changes than Fv /Fm . The wheat material of 41 cultivars used in this study has previously been selected among 1274 original cultivars during three rounds of screening to identify cultivars significantly different in Fv /Fm when challenged by heat stress on intact plants (Sharma et al., 2012). The relationship between the responses of intact plant versus detached leaf of the same 41 cultivars was studied to test whether a cultivar with a high/low Fv /Fm in the intact plant treatment showed a correspondingly high/low Fv /Fm in the detached leaves. However, this was not the case because the response of the cultivars did not correlate in the two methods. Two separate experiments (expt.1 and expt.2) with detached leaves treatment at the same temperature and duration in a water bath (40 ◦ C for 30 min) did produce a similar genotypic response to some extent, indicating that genetic differences do exist for the detached leaf treatment. However, such correlation of the genotypic response disappeared in method 1 versus method 2, indicating genetic differences affecting responses in the two methods. A possible role of differences in growth conditions in the two methods looks unlikely because the cultivar Fv /Fm in two experiments/two methods (because expt.2 was conducted at the same time as intact plant experiment) before the start of the treatment was similar, as indicated by a significant positive correlation. Therefore, the differences in genotypic response are caused by the treatment, but the genetic difference among cultivars was not expressed in a similar manner in the two methods. Comparing genetic determination of chlorophyll fluorescence parameters on intact plants versus detached leaves, we found that the genetic differences among cultivars decreased under heat stress conditions in detached leaves, in contrast to results from intact plants (Sharma et al., 2012). It is also important to note that measurements were carried out in a similar way in both intact and detached leaf methods, and the only difference was the way plant

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material was heat stressed. In the present study, the chlorophyll fluorescence parameters remained stable at 20 ◦ C (control) with all of the durations, and the stress effect could not be attributed to a wounding or detachment effect of the leaves. The use of detached leaves to probe the cold stress response has been questioned, as it altered the ranking of the species for cold tolerance (Kaniuga et al., 1978). On the other hand, it has also been suggested in chilling experiments that both attached and detached leaves would behave comparably if the similarity in the experiment were well preserved (Smillie et al., 1987). A leaf fragment system has been reported to be an efficient method for detection of water stress tolerance in castor bean cultivars based on electrolyte leakage and chlorophyll fluorescence (Faria et al., 2013). Furthermore, Srinivasan et al. (1996) evaluated the heat tolerance in legumes by cell membrane thermo stability and the chlorophyll fluorescence technique using both in vivo and in vitro tests, which largely yielded similar results in ranking of legumes. However, it was also reported that this method is not suitable for studying recovery and whole plant mechanisms during heat stress. In the present study, intact plant and detached leaf methods yielded different genotypic responses under heat stress. This may not imply that the detached leaf treatment cannot be used for selection of heat tolerant wheat cultivars. Instead, the results show that the genetic differences among cultivars affecting the two stress treatment methods might not be the same. It is important to be aware that this wheat material was selected for Fv /Fm under heat stress conditions using intact plants. If the entire selection process to identify wheat cultivars differing in heat tolerance is done with the detached leaf method, it may be possible to identify a new set of discriminating cultivars, possibly with similar/dissimilar sets of genes affecting tolerance to the heat stress imposed on detached leaves. Nevertheless, it is generally the integrated response mechanisms in whole plants that allow them to endure stress conditions. In conclusion, although the detached leaf method offered advantages for screening large numbers of genotypes under heat stress by saving resources and avoiding confounding effects of other stress factors in the treatment, the cultivar response of detached leaves to high temperature does not seem to be the same as in intact plants. The main difference between the two methods is that the heat-induced effects occur at a much faster rate in detached leaves, which may be due to the integrated physiological potential of the intact plant versus physiochemical processes alone in detached leaves. Acknowledgements This work was funded by the project ‘HeatWheat’ (Grant Number 3304-FVFP-09-B-008) from the Food Research Program 2009 of the Ministry of Food, Agriculture and Fisheries, Denmark. The authors also acknowledge ‘Erasmus Student Mobility Program’ for supporting Juan Olivares Fernández during his internship in the University of Copenhagen, Denmark. References Ali MB, Ibrahim AMH, Hays DB, Ristic Z, Fu J. Wild tetraploid wheat (Triticum turgidum L.) response to heat stress. J Crop Improv 2010;24:228–43. Baena-Gonzalez E, Aro EM. Biogenesis, assembly and turnover of photosystem II units. Philos Trans R Soc Lond Ser B Biol Sci 2002;357:1451–9. Baker NR, Rosenqvist E. Applications of chlorophyll fluorescence can improve crop production strategies: an examination of future possibilities. J Exp Bot 2004;55:1607–21. Bonos SA, Murphy JA. Growth responses and performance of Kentucky bluegrass under summer stress. Crop Sci 1999;39:770–4. Box GEP, Cox DR. An analysis of transformations. J R Stat Soc Ser B Stat Methodol 1964;26:211–52. Demmig B, Björkman O. Comparison of the effect of excessive light on chlorophyll fluorescence (77 K) and photon yield of O2 evolution in leaves of higher plants. Planta 1987;171:171–84.

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